Synaptic transmission depends on the storage of neurotransmitter within vesicles that undergo regulated exocytosis. However, previous studies have suggested the storage of different classical transmitters in distinct vesicular compartments. To understand the mechanisms responsible for storage in different secretory vesicles and its biological role in synaptic transmission and behavior, the program focuses on the localization of membrane proteins involved in the transport of classical neurotransmitters into secretory vesicles. Recent molecular cloning of two vesicular monoamine transporters (VMATs) and a closely related vesicular acetylcholine transporter (VAChT) has enabled us to determine the subcellular localization of these proteins. In PC12 cells as well as in brain, the VMATs localize preferentially to large dense core vesicles (LDCVs) whereas VAChT localizes to small synaptic vesicles (SVs). In the first specific aim, we will identify the signals responsible for sorting VMAT2 to LDCVs. If the signals occur in a cytoplasmic domain, we will use them to isolate components of the cytosolic sorting machinery. The work thus addresses the biogenesis of LDCVs (that contain neural peptides and growth factors) as well as the site of monoamine storage. In the second specific aim, we will characterize the vertebrate homologue of a C. elegans gene implicated in vesicular GABA transport. Consistent with the differences in bioenergetics from the VMATs and VAChT, the sequence defines a novel gene family that may also include the vesicular transporter for glutamate and we have isolated two related vertebrate sequences. We will now determine the distribution of the different sequences and characterize the functional properties of the proteins they encode. The results will indicate whether cell populations release multiple classical transmitters and enable us to assess the role of these transport activities in forms of neural plasticity such as long-term depression and potentiation. The third specific aim addresses the function of VMAT2 in vivo by targeting the disruption of the mouse gene. Using the homozygous mice, we will assess the role of VMAT2 in development and in degeneration. We will also use the mice as a null background to introduce mutant forms of VMAT2 that are defective in either transport or localization. In particular, localization of VMAT2 to SVs rather than LDCVs would help to determine the significance of storage in these different vesicle types on behavior.